Recombinant DNA technology enables the production of genetically altered plants through the introduction of preconstructed exogenous genes or “transgenes” in random, atopic positions. By contrast, the present invention makes it possible to make a specific alteration of a specific target gene of a plant through the use of MDON. We have developed improved methods of introducing MDON in to plant cells in order to achieve specific modifications of a target gene while improving the recovery of viable, fertile plants that have the desired genetic modifications.
Use of MDON to Effect Specific Genetic Alterations
Mixed duplex oligonucleotides (MDON) and their use to effect genetic changes in eukaryotic cells are described in U.S. Pat. No. 5,565,350 to Kmiec (Kmiec I). Kmiec I discloses inter alia MDON having two strands, the first strand containing two segments of at least eight RNA-like nucleotides that are separated by a third segment of from 4 to about 50 DNA-like nucleotides, termed an “interposed DNA segment.” The nucleotides of the first strand are base paired to DNA-like nucleotides of a second strand.
The first and second strands are additionally linked by a segment of single stranded nucleotides, so that the first and second strands are parts of a single oligonucleotide chain. Kmiec I further teaches a method for introducing specific genetic alterations into a target gene. According to Kmiec I, the sequences of the RNA segments are selected to be homologous, i.e., identical, to the sequence of a first and a second fragment of the target gene. The sequence of the interposed DNA segment is homologous with the sequence of the target gene between the first and second fragment except for a region of difference, termed the “heterologous region.” The heterologous region can effect an insertion or deletion or can contain one or more bases that are mismatched with the sequence of target gene so as to effect a substitution. According to Kmiec I, the sequence of the target gene is altered as directed by the heterologous region, such that the target gene becomes homologous with the sequence of the MDON. Kmiec I specifically teaches that nucleotides that contain ribose and 2′-O-methylribose, i.e., 2′-methoxyribose, can be used in MDON and that naturally-occurring deoxyribose-containing nucleotides can be used as DNA-like nucleotides.
U.S. Pat. No. 5,731,181 (Kmiec II), discusses the use of MDON to effect genetic changes in plant cells and provides further examples of analogs and derivatives of RNA-like and DNA-like nucleotides that can be used to effect genetic changes in specific target genes.
Scientific publications disclosing uses of MDON having interposed DNA segments include Yoon, et al., 1996, Proc. Natl. Acad. Sci. 93:2071-2076 and Cole-Straus, A. et al., 1996, Science 273:1386-1389. The rates of mutation achieved in these studies ranges to as high as about one cell in ten using liposome-mediated delivery. However, these publications do not disclose that MDON can be used to make genetic changes in plant cells.
Introduction of MDON into Plant Cells using Electroporation and Microprojectile Bombardment. Kmiec I and II discuss the use of electroporation for introduction of MDON into plant protoplasts. The regeneration of fertile plants from protoplast cultures has been reported for certain species of dicotyledonous plants, e.g., Nicotiana tobacum (tobacco), U.S. Pat. No. 5,231,019 and Fromm, M. E., et al., 1988, Nature 312, 791, and soybean variety Glycine max, WO 92/17598 to Widholm, J. M. However, despite the reports of isolated successes using non-transformed cells (Prioli, L. M., et al., Bio/Technology 7, 589; Shillito, R. D., et al., 1989, Bio/Technology 7, 581), the regeneration of fertile monocotyledonous plants from transformed protoplast cultures is not regarded as obtainable with application of routine skill. Frequently, transformed protoplasts of monocotyledonous plants result in non-regenerable tissue or, if the tissue is regenerated, the resultant plant is infertile.
U.S. Pat. Nos. 4,945,050, 5,100,792 and 5,204,253 concern microprojectile bombardment, the delivery of plasmids into intact plant cells by adhering the plasmid to a microparticle that is ballistically propelled across the cell wall, hereafter “biolistically transformed” cell. For example U.S. Pat. No. 5,489,520 describes the regeneration of a fertile maize plant from a biolistically transformed cell.
U.S. Pat. No. 5,302,523 discusses the introduction of plasmid DNA into suspensions of plant cells having intact cell walls through the use of silicon carbide fibers that pierce the cell wall.
U.S. Pat. No. 5,384,253 discusses the use of a combination of endopectin lyase (E.C. 3.2.1.15) and endopolygalacturonase (E.C. 4.2.2.3) to generate transformation-competent cells that can be more readily regenerated into fertile plants than true protoplasts. However, the technique is reported to be useful only for F1 cell lines from the cross of line A188×line B73.
Mutagenesis of Plant Genes to Confer Herbicide Tolerance. U.S. Pat. No. 4,535,060 discusses the production of herbicide-tolerant plants by introducing mutations into plant genes that encode certain enzymes that when mutated are more resistant to a competitive inhibitor of the enzymes that acts as an herbicide. However, results from these techniques have been limited, and more reliable methods with wider applicability are needed.
The possibility of gene replacement in plants by homologous recombination (the Smithies-Capecchi technique) has been discussed, but this technique has not been successfully applied to plants.
Imidazolinone resistance can be conferred on plants by mutations in the aminohydroxy acid synthase (AHAS) gene. This is exemplified by “Smart Canola,” which has a mutation in AHAS 1 gene at amino acid position 635 (also known as PM1) and a mutation in AHAS 3 at the same amino acid position (PM2). The PM2 mutation also confers resistance to an additional family of herbicides.